Immunoassay utilizing monoclonal high affinity IgM antibodies

ABSTRACT

Hybridomally produced monoclonal IgM antibodies having high affinity are useful for the immunoassay and purification of viral antigens.

BACKGROUND OF THE INVENTION

The invention described herein was made in the course of work under a grant or award from the Department of Health and Human Services.

1. Field of the Invention

The present invention relates to an immunoassay system which utilizes hybridomally produced high affinity IgM antibodies.

2. Description of the Prior Art

The use of immunoassay for the detection of minute amounts of substances in physiological fluids is very well known in the art. The assay basically depends on the binding interaction between an antigen and an antibody therefor. In the great majority of systems described in the prior art, the antibody is usually immunoglobulin G (IgG). Among these, the polyclonal IgG's have until recently been routinely utilized (see for example T. Chard, "An Introduction to Radioimmunoassay and Related Techniques", North-Holland Publishing Company, 1978).

The advent of hybridomal techniques has brought about the possibility of producing homogeneous populations of highly specific antibodies against a variety of antigens. Thus, Koprowski et al U.S. Pat. No. 4,172,124 describe antibodies demonstrating specificities for malignant tumors produced by somatic cell hybrids between myeloma cells and spleen or lymph cells. Koprowski et al U.S. Pat. No. 4,196,265 describe continuous cell lines of genetically stable fused cell hybrids capable of producing large amounts of IgG antibodies against specific viruses, such as influenza virus, rabies, mumps, SV40, and the like. Zurawski et al, Federation Proceedings 39:4922 (1980), disclose hybridomas producing monoclonal IgG antibodies against tetanus toxin. Frankel and Gerhard, Molecular Immunology, 16:101-106 (1979) also disclose monoclonal antiviral hybridoma IgG antibodies described as "true bivalent antibodies", Frankel and Gerhard, at 105. The Frankel and Gerhard paper describes what are probably the prior art antibodies having the highest affinities to date. The monoclonal antibodies of Frankel and Gerhard have K values (binding constants determined from Scatchard plots) in the ranges of from less than 10⁵ M⁻¹ to about 10¹⁰ M⁻¹. The aforementioned Zurawski et al paper describes monoclonal antibodies having K association constants in the range of 3.5-5.4×10⁸ M⁻¹.

The use of IgM antibodies has received relatively less attention in the immunoassay art. IgM antibodies consist of 5 units each the approximate size of IgG joined by a so-called J (joining) chain. The IgM molecule has a valency of 10, although quantitative antigen binding measurements often detect only 5. IgM's are extremely sensitive to reducing agents, they self-aggregate readily and precipitate out of solution, and have been found to bind in a non-specific manner. These reasons, among others, have heretofore prevented their extensive use. Because of the polyvalency of IgM antibodies, various suggestions have been made in the art concerning the theoretical binding affinity of these antibodies towards antigens. For example Metzger, has suggested that it would be "intuitively obvious" that an antibody with multiple binding sites would bind more firmly than a univalent or bivalent antibody to a multivalent antigen. Metzger however qualifies this statement recognizing that the energetics of binding would be highly dependent on the number and topology of antigenic determinants, the distribution of combining sites in space and the balance between the free energy of binding released by combining site/determinant interactions and high free energy required to maximize the number of multiple flexible conformations existing in such large molecules. These qualifications are even more stringent when it comes to cross linking of translationally independent determinants such as in agglutination. Mathematical models have been developed describing the theoretical binding of multivalent IgM antibodies to multideterminant antigen particles (Crothers and Metzger, Immunochemistry, Volume 9, 341-357 (1972)). It has even been suggested (Ehrlich, Journal of Theoretical Biology, 81:123-127 (1979)) that multivalency might theoretically increase specificity. All of these expectations of the art, as represented by the intuitive feelings and purely theoretical mathematical models, have been at odds with the experimental facts except in certain specific instances, infra. Although IgM antibodies are multivalent, the binding affinities observed for them have, prior to this invention, always been substantially less than the binding affinities for IgG antibodies. It is for this reason that IgM's have not routinely been utilized for immunoassays, other than in certain specific circumstances, infra. The art has explained the observed poor binding affinity of IgM antibodies, as being due to the structural features of the molecules, and the probability that combining sites of IgM molecules have lower avidity for antigens than do those of IgG's. Cunningham, A. ("Understanding Immunology", Academic Press Inc., 1978, at 33), in suggesting why quantitative antigen binding measurements with larger antigens on IgM antibodies detect only 5 and not 10 binding sites, discusses the possibility that for reasons of steric restriction, all the sites cannot attach to the same antigenic structure at the same time.

Faced with the lesser utility of IgM antibodies in conventional precipitation or immunoassay techniques, the prior art nevertheless details some special circumstances under which this class of antibodies can, and has been used. Soothill et al U.S. Pat. No. 4,210,622 describe the use of heterologous low affinity IgM obtained from myeloma serum for the selective determination of immune complexes. This technique takes advantage of the fact that low affinity IgM's will selectively bind antibody-antigen complexes even in the presence of individual components of the complex. Soothill et al U.S. Pat. No. 4,141,965 describe a method wherein low affinity heterologous IgM antibodies are used to agglutinate antigen-coated latex particles. When physiological fluid containing antigen is added to the agglutinate mixture, an inhibition-of-agglutination occurs. Gopalakrishnan and Karush, Journal of Immunology, 113: 769-778 (1974), disclose the use of IgM antibodies of restricted heterogeneity induced by a lactoside determinant, for the binding of conjugates of the lactoside determinant with bacteriophage φX174. These authors report that the constant for the binding of the antibody to the conjugate (˜10⁹ M⁻¹) is greater by a factor of 10,000 than that for the lactoside itself (˜10⁵ M.sup. -1). Blank et al, Journal of Immunology, 108:665-673 (1972)) disclose affinity constants for grouper IgM's in the order of 10⁴ M⁻¹. A specific instance of an IgM antibody, rheumatoid factor (RF) has also been utilized for immunoassays in the past. RF's are IgM antibodies directed towards antigenic determinants present on autologous immunoglobulin. Lurhuma et al, Clinical and Experimental Immunology, 25:212-226 (1976), describe the use of RF as an agglutinant for IgG coated particles, and an inhibition of agglutination effect which occurs in the presence of antibody-antigen complexes. Masson et al U.S. Pat. No. 4,062,935 describe a similar system to that of Lurhuma.

Monoclonal RF to human IgG has been used by Eisenberg, Immunochemistry, 13:355-359 (1976) to demonstrate binding to aggregated IgG. The binding of monoclonal RF to aggregated IgG is increased by a factor of greater than 10⁶ (as determined by radioimmunoassay) over the binding of RF to nonaggregated, monomeric, IgG.

In sum, the use of IgM antibodies, whether polyclonal or monoclonal has been restricted to assaying for antibody-antigen complexes, aggregated IgG's, or, by an inhibition of agglutination technique, to antigen-coated latex particles. Because of the poor binding affinity of IgM's as well as their self-aggregating tendency, non-specific interactions and sensitivity to reducing agents, it has generally been assumed in the art that IgM antibodies were not useful for highly sensitive immunoassay techniques.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a highly sensitive immunoassay system for viral antigens.

Another object of the invention is to provide an immunoassay system utilizing IgM antibodies.

These and other objects of the invention as will hereinafter become more readily apparent have been attained by providing:

An immuoassay system utilizing an antibody to assay for a viral antigen, wherein said antibody is a hybridomally produced monoclonal high affinity IgM.

Another object of the invention is to provide solid phase bound hybridomally produced IgM antibodies useful for immunoassay techniques and for purification methods.

Yet another object of the invention is to provide a purification method for viral antigens which utilizes solid phase bound hybridomally produced IgM antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

FIG. 1 shows the purification of hepatitis B surface antigen (HBsAg) of the adw subtype from serum, with a hybridomally produced monoclonal IgM anti-HBs affinity column. The elution profile of protein and HBsAg activity is measured by a solid phase radioimmunoassay from a cyanogen bromide activated Sepharose 6B control column.

FIG. 2 shows a similar purification to that of FIG. 1 wherein the elution profile is from a control column prepared with 30 mg of mouse serum.

FIGS. 3 and 4 represent affinity columns prepared with 28 and 65 mg of ascites fluid protein from clones 5D3 and 2F11. It should be noted that the monoclonal hybridomally produced IgM anti-HBs coupled to Sepharose 6B bound HBsAg, and substantial antigenic activity and protein were recovered following elution with glycine-HCl pH2.6. Negative serum controls for HBsAg activity where 426 106 cpm and positive serum controls were 12,462±321 cpm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Wands and Zurawski, copending U.S. patent application Ser. No. 086,947 filed Oct. 22, 1979 at the U.S. Patent Office, now U.S. Pat. No. 4,271,145 and which is in its entirety herein incorporated by reference, discloses a process for hybridomally producing monoclonal IgM antibodies against a variety of antigens, especially viral antigens. The present invention arose out of the suprising discovery that the thus produced monoclonal IgM antibodies, not only show the homogeneous population specificity which is expected in hybridomally produced antibodies but, in addition, show unexpectedly high binding affinity constants for viral antigens. Although the art had intuitively predicted and theoretically modeled the possibility that IgM antibodies might show increased binding affinities, most experimental results obtained prior to this invention, as discussed previously, did not bear out these predictions, and even if they did to some extent, were not sufficiently useful for sensitive immunoassay techniques. The actually observed binding constants for polyclonal IgM antibodies have generally been in the range of 10⁴ -10⁶ M⁻¹. It was assumed prior to this invention that a certain variation in affinity constants existed in any population of IgM antibodies. It was not known however how high the affinity constants could be, and none had been observed to be higher than about 10⁹ M⁻¹. The present inventors have discovered that hybridomally produced antibodies against viral antigens can have affinity binding constants higher than 10⁹ M⁻¹ and as high as 10¹¹ M⁻¹.

Through the use of monoclonal hybridomally produced IgM antibodies, the present inventors have now simultaneously improved the specificity and the affinity of immunoassays, in all reactions between IgM antibodies and viral antigens. By increasing the affinity binding constant to the high values observed in the present invention, it is possible for the first time to carry out highly sensitive immunoassay procedures using IgM antibodies. The combined use of a hybridomally produced IgM antibody and a viral antigen allows the improvement in performance.

The specificity relates to the lack of cross reactivity or lack of interference in the antigen-antibody reaction by other substances present in the reaction mixture. Hybridomally produced antibodies in general, including IgG's, exhibit an extraordinary specificity since they are directed against a single determinant. However because they are directed against a single determinant, they bind to fewer sites on an antigen than classically (polyclonally) produced antibodies. This fact had been reflected in an existing fear in the prior art that the extraordinary specificity of monoclonally produced antibodies, would be a detriment to their utilization in sensitive clinically useful immunoassays. The present inventors however have now demonstrated that hybridomally produced IgM's can detect less antigen than the conventionally produced antibodies, despite their extraordinary specificity.

The hybridomally produced IgM antibodies for specific viral antigens utilized in the present invention can be obtained, for example, through the use of cell fusion techniques first described by Kohler and Milstein (Nature, 255:495 (1975)). Lymphocytes from an animal immunized with the antigen of choice are fused to a continuously proliferating myeloma cell. Subsequently, hybrid cells are selected in hypoxanthine-aminopterin-thymidine (HAT) medium (Littlefield, Science, 145:709 (1964)). Those which produce the IgM antibody of choice are cloned and propagated indefinitely in tissue culture or in syngeneic or immunocompromised animals, or in athymic animals, for example nude mice. Such clones, are the progeny of single cell produced or monodisperse IgM antibodies, i.e., monoclonal IgM antibodies, which is directed to an individual determinant or an antigen. This determinant is expressed more than once on the surface of each individual viral particle or fragment thereof. Further details on the preparation of monoclonal IgM antibodies can be found in the aforementioned Wands and Zurawski U.S. Pat. No. 4,271,145.

Analysis of Hybridomas. FIG. 5 illustrates the results from one successful fusion as described in the Wands and Zurawski application, under optimal immunization conditions and depicts the range of anti-HBs activity in 47 positive cell lines. The areas between the parallel dotted lines in this and the other figures represents the means±SEM of the negative controls. In this experiment, assays for anti-HBS binding using the [¹²⁵ I]-HBsAg probe were performed 10 to 20 days after the initial fusion when growth was observed in the 96 well microtiter plates. It is noteworthy that 120 μl of cell culture supernatant (total volume 200 μl/well) obtained from some hybridomas yielded extraordinarily high values for [¹²⁵ I]-HBsAg binding activity. For example, anti-Hbs produced by cell line 2E4 bound 95,000 of the 100,000 cpm added in the radioimmunoassay. Table la shows Anti-HBs activity of hybridoma cell lines 1F8, 2F11 and 5D3 following dilution.

                  TABLE 1a                                                         ______________________________________                                         Anti-HBs activity of hybridoma cell lines 1F8, 2F11, and                       5D3 following dilutional plating.                                                   No.      No. wells    cpm [.sup.125 1]-HBsAg bound                        Cell of wells pos.         (mean ± SEM)                                     line seeded   anti-HBs 10.sup.4*                                                                          10.sup.3 10.sup.2                                   ______________________________________                                         1F8  60       60    29.3 ± 0.71                                                                          28.9 ± 1.06                                                                          19.36 ± 1.74                          2F11 60       60    26.42 ± 0.43                                                                         26.12 ± 0.63                                                                         13.86 ± 0.80                          5D3  60       60    88.98 ± 1.64                                                                         99.99 ± 3.26                                                                         66.07 ± 3.50                          ______________________________________                                           *The number of cells seed/well                                                  cpm [.sup.125 1HBsAG × 10.sup.-3 bound                           

Cloning of Hybridomas. FIG. 6 is a representative example of the dilutional cloning of line 5D3. Of initial 120 wells seeded at 0.5 cells/well, 53 or 44% yielded positive hybridoma cell growth; only 21 of the 53 gave anti-HBs binding values greater than 1000 cpm and were considered positive anti-HBs secretors. It is noteworthy that 120 μl of culture supernatant obtained from 12 such cell lines produced anti-HBs which bound more than 50,000 of the 100,000 cpm of added [¹²⁵ -I]-HBsAg; these values were in excess of those obtained with hemophiliac sera. Two of the hybridomas with high anti-HBs activity (100,000 and 96,000 cpm [¹²⁵ I]-HBsAg bound respectively) were recloned by the same technique and all showed 95-98% binding value.

Other techniques of production might of course also be used, such as viral transformation, recombinant DNA and other gene splicing or manipulation techniques.

The hybridomally produced high affinity IgM antibodies of the present invention are those having affinity binding constants greater than 10⁹ M⁻¹, preferably greater than 10¹⁰ M⁻¹, most preferably greater than 10¹¹ M⁻¹. In order to produce such high binding antibodies it is necessary to use a viral antigen or a fragment thereof.

Among the viral antigens which may be determined in the present invention are virus particles, fragments of virus particles such as whole coats, or individual molecular components of the coat, e.g., polypeptides or lipid or carbohydrate complexes. Examples of viral antigens are hepatitis B surface antigens, hepatitis Be antigen, hepatitis B core antigens, hepatitis A virus and isolated antigens thereof, herpes viruses I and II, herpes virus Zoster, cytomegalovirus, Epstein-Barr virus, papova viruses such as BK or JC virus, measles virus, rubella virus, influenza or para influenza virus, or any other viruses known or suspected of being etiologic in pathogenesis or animals or plants.

The high affinity hybridomally produced IgM antibodies of the present invention can be used in any of the array of available immunoassay techniques which utilize the binding interaction between an antibody and an antigen, and the present invention is not limited to any of these techniques. The most common of these is radioimmunoassay (RIA). RIA is a well known technique and will not be described in detail here. For particulars, reference is made to Chard, "An Introduction to Radioimmunoassy and Related Techniques", North-Holland Publishing Company, 1978. Any of the many variations of RIA can be used, such as homogeneous phase RIA, or heterogeneous or solid phase RIA, single antibody methods or "double" antibody methods, and direct (forward) or reverse sandwich assays. Particularly preferred are solid phase systems wherein the antibody (IgG or IgM) is covalently coupled to an insoluble support so that both the antibody and the bound complex after incubation can be readily separated from the soluble free fraction. A wide variety of solid phase supports have been described, which include particles of dextran, cellulose, continuous surfaces such as polystyrene or polypropylene discs, walls of plastic tubes, glass discs, glass particles, and the like. Particulate solid phases are widely used for a variety of different assays and are included in the present invention. Antibodies are attached to the particles by any of a number of techniques designed to yield a nonreversible covalent or non-covalent link between protein and particle, for example directly or by cyanogen bromide activation. Other alternatives are the use of antibodies entrapped in the interstices of a polyacrylamide gel, or bound to magnetic particles. The assay tube is set up with sample or standard, tracer and the appropriate amount of solid phase bound antibody, plus a detergent to prevent aggregation of the particles and non-specific absorption of the tracer. After an incubation period during which the tubes are continuously mixed, the solid phase is sedimented by centrifugation; the supernatant is removed and the solid phase subject to two or more washes with buffer in order to remove free tracer trapped within and between the particles. The counts on the solid phase (bound fraction) are then measured. Immunoradiometric assays, Chards, supra at 423, can also be used. When a second antibody radioimmunoassay system is used, the second antibody may be IgM or may be IgG.

Another immunoassay technique useful with the IgM antibodies of the present invention is enzyme immunoassay. This technique is also well known to the art and reference is made to Schuurs and VanWeemen, Clinica Chimica Acta, 81:1-40 (1977). In this technique, enzymes are applied as labels on antigen or antibodies for identification and localization of the immunoreactants. Any method in which the extent of binding of enzyme-labeled antigen, or enzyme labeled-antibody to its immunoreactant is measured, is included in this invention. Enzyme immunoassays can be classified as homogeneous or heterogeneous, depending on whether the labeled reagent behaves differently or identically whether or not it is bound to specific counterparts in the immunoreaction, and which therefore may or may not require physical separation of the reactants into two fractions. The variety of enzymes used, methods of linking enzymes to the immunological components, purification of the conjugates, as well as various assay principles and methods are well described in the aforementioned Schuurs and VanWeemen article. Needless to say, any enzyme immunoassay which has used IgG antibodies in the past can be used with the high affinity IgM antibodies of the present invention.

Another immunoassay method useful in the present invention is the latex agglutination method. In this method, latex particles are coated with viral antigen and incubated with high affinity hybridomally produced IgM antibodies. Inhibition of agglutination will occur when a sample of physiological fluid containing the viral antigen is incubated with this mixture. The inhibition of agglutination can either be followed with a counter or by recently developed infrared absorption techniques. An alternative is to coat the latex particles with the hybridomally produced IgM antibodies. Incubation of these coated particles with physiological fluid comprising viral antigen will cause agglutination. Instead of latex particles, animal cells such as red blood cells can of course be used. In this case, the technique becomes a variation of the well known hemagglutination technique used with IgG antibodies and red blood cells.

Other useful immunoassay techniques are those using other labeling techniques such as:

fluorescent dyes (Aalbeses, R. C. Clin. Chim. Acta 48:109-111 (1973);

electron-dense compounds (ferritin) Singer, S. J. et al, J. Biophys. Biochem. Cyto; 9: 519-537 (1961);

protein - bacteriophage conjugates, (Haimovich, J. et al, Biochim. Biophys. Acta 207: 115-124 (1970); or

stable free radicals (Bastiani, R. J., et al, Am. J. Med. Technol. 39: 211-216 (1973).

Because of the high affinity and specificity of the monoclonal IgM antibodies of the present invention it is also possible to use the same in purification methodologies. The most common of these comprises binding the hybridomal IgM antibody on a cellulose based solid phase support, adding a complex mixture containing the viral antigen recognizable by the antibody, thereby binding said antigen to said antibody, washing the column extensively with buffers and eluting the viral antigen by changing the column conditions, such as the ionic strength, the pH or the like.

Having now generally described this invention, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless specified.

EXAMPLE 1

This example describes the use of IgM monoclonal antibodies to hepatitis B surface antigen (HBsAg) in several solid phase radioimmunoassays. Two general types of radioimmunoassays are defined. One assay is performed in the reverse direction. Under these experimental conditions serum containing the hepatitis antigen is first incubated with ¹²⁵ I-monoclonal IgG or IgM antihepatitis B antigen, or heterologous antihepatitis B (prepared by conventional immunizations) followed by a second incubation with monoclonal IgM anti HBs or heterologous anti-HBs coupled to a solid phase, for example polystyrene beads. The other assay is performed in the forward direction. In this protocol, monoclonal IgM anti-HBs or heterologous anti-HBs coupled to a solid phase is first incubated with serum containing HBsAg. The second incubation, therefore, is comprised of ¹²⁵ I-monoclonal IgG or IgM anti-HBs or heterologous anti-HBs. The sensitivity and specificity of the monoclonal IgM and IgG anti-HBs sandwich assays are compared directly to conventional assays either alone or in combination with anti-HBs prepared by standard animal immunizations.

MATERIALS AND METHODS

Monoclonal IgM and IgG antibodies with specificity for HBsAg were obtained according to the procedures described by Wands and Zurawski copending U.S. patent application No. 086,947, Oct. 22, 1979, now U.S. Pat. No. 4,271,145. One quarter inch plastic beads were obtained commercially; identical beads coated with conventionally prepared antibodies to HBsAg (AUSRIA II) were also obtained commercially (Abbott Laboratories, N. Chicago, Ill.)

Plastic beads were coated with IgM hybridomal anti-HBs as follows: Ascites fluid derived from malignant growth in BALB/mice of double cloned hybridoma cell lines which produce IgM anti-HBs antibodies was diluted 1:1000 to 1:5000 in phosphate buffered saline. The beads were incubated for 16 hours at room temperature and washed X3 with distilled water: the beads coated with IgM anti-HBs were then ready for use in the RIAs. The HBsAg was radiolabeled with Na¹²⁵ I using the chloramine T method of Hunter and Greenwood, Nature 194:495 (1962) or Hunter and Bolton, Biochemical Journal, 133: 529-(1973) ; specific activity ranged from 0.1 to 0.4 μCi/μg and incorporation of ¹²⁵ I was 20-26 percent. The monoclonal IgG anti-HBs antibodies were also labeled according to the above method; specific activity ranged from 0.1 to 15 μCi/μg and incorporation of radiolabel varied from 20 to 50 percent.

Assays were carried out with various combinations of coated beads and labeled antibodies or antigen to establish the efficacy of the technology. In general individual coated beads were washed X3 with distilled water then mixed with dilutions of patient sera, HBsAg standards, radiolabeled HBsAg or anti-HBs as described in the results. Following washing, the beads were reincubated with radiolabeled reagents as necessary, washed, collected and counted in a Packard gamma well spectrometer. Incubation time varied between 1 and 16 hours as described below.

RESULTS

Table 1b represents the kinetic binding data obtained when plastic beads coated with monoclonal IgM anti-HBs (designated 5D3 and 1F8) were incubated with labeled ¹²⁵ I-HBsAg.

                                      TABLE Ib                                     __________________________________________________________________________     Comparison of .sup.125 I-HBsAg binding between monoclonal IgM anti-HBs         coated beads and beads coated with conventionally prepared guinea              pig anti-HBs (AUSRIA II)                                                       Time                cpm bound*                                                 (hrs.)                                                                            AUSRIA II                                                                             %**                                                                               5D3+   %      1F8    %                                            __________________________________________________________________________     1  20,286 ± 412                                                                       8.1                                                                               15,261 ± 106                                                                       12.0   13,216 ± 940                                                                       10.4                                         2  15,731 ± 621                                                                       12.4                                                                              22,916 ± 310                                                                       18.2   19,419 ± 612                                                                       15.4                                         3  22,216 ± 491                                                                       17.6                                                                              31,916 ± 290                                                                       25.3   29,312 ± 416                                                                       23.3                                         4  26.219 ± 301                                                                       20.8                                                                              45,000 ± 216                                                                       35.7   42,101 ± 601                                                                       33.4                                         7  35,109 ± 720                                                                       27.8                                                                              55,109 ± 690                                                                       43.7   54,469 ± 888                                                                       43.2                                         12 42,509 ± 612                                                                       33.7                                                                              62,916 ± 94                                                                        49.9   60,001 ± 714                                                                       47.6                                         16 48,216 ± 819                                                                       38.2                                                                              75,928 ± 110                                                                       60.3   --     --                                           24 57,016 ± 126                                                                       45.2                                                                              89,912 ± 430                                                                       70.5   --     --                                           __________________________________________________________________________      *126,000 cpm .sup.125 IHBsAg added to coated beads                             **Percent of total cpm bound                                                   + 5D3 is a monoclonal IgM antiHBs with specificity for adw and ayw             subtypes                                                                         1F8 is a monoclonal antibody with specificity primarily to adw subtype 

It is evident that the two monoclonal IgM coated beads bind HBsAg at a faster rate than the conventional AUSRIA II IgG anti-HBs antibodies. Moreover, a greater percent of total ¹²⁵ I-HBsAg added is bound at 24 hours by the IgM antibodies. A simple substitution was made of the 5D3 coated beads for the AUSRIA II coated beads and the sensitivity of the two assays was directly compared as shown in Table II. In this experiment dilutions of 2 patients' sera known to contain HBsAg were incubated with 5D3 coated beads or AUSRIA II beads for 16 hours. The beads were washed X3 with distilled water. 200 μl (150,000 cpm) of AUSRIA II ¹²⁵ I-guinea pig antibodies (anti-HBs) prepared by conventional immunization were then added to both sets of beads for 4 hours. The beads were washed again and counted.

                                      TABLE II                                     __________________________________________________________________________     Improvement in the AUSRIA II radioimmunoassay for measurement of               HBsAg in serum by substitution of monoclonal IgM anti-HBs coated               beads for the AUSRIA II beads                                                               Mean cpm*                                                                             Mean cpm**                                                                            S/N.sup.+                                                                             S/N.sup.+                                    Patient                                                                             Serum dilution                                                                         AUSRIA II                                                                             5D3    AUSRIA II                                                                             5D3                                          __________________________________________________________________________     1    1 × 10.sup.4                                                                     5,666  16,308 35     130                                               1 × 10.sup.5                                                                     2,849  4,289  18     34                                                2 × 10.sup.5                                                                     2,145  3,084  13     27                                                4 × 10.sup.5                                                                     1,249  1,857  7.7    15                                                8 × 10.sup.5                                                                     819    1,144  5.5    9.1                                               16 × 10.sup.5                                                                    512    615    3.5    6.0                                          2    1 × 10.sup.4                                                                     6,061  14,883 38     119                                               1 × 10.sup.5                                                                     2,652  4,486  16     36                                                2 × 10.sup.5                                                                     1,810  2,388  11.2   19                                                4 × 10.sup.5                                                                     1,249  1,679  7.8    13                                                8 × 10.sup.5                                                                     774    893    4.8    8                                                 16 × 10.sup.5                                                                    625    545    3.9    5                                            Negative                                                                       Controls                                                                            --      161    125                                                        __________________________________________________________________________      *AUSRIA II beads incubated × 16 hr with 200 μl of patients serum      washed 3× with distilled water followed by a 4 hour incubation with      .sup.125 Ianti-HBs conventionally prepared in guinea pig and washed            ×3 in distilled water. Beads counted in gamma counter.                   **Procedure and reagents identical to above except 5D3 coated beads            substituted for AUSRIA II beads. +S/N is the signal/noise calculated by        dividing the mean cpm in test sample by mean cpm of the negative controls                                                                               

It is evident from Table II that a simple substitution of the 5D3 coated beads improved the AUSRIA II assay at both high and low HBsAg concentrations. Furthermore, the background counts exhibited by the negative control sera was lowered by substituting the 5D3 IgM coated beads for AUSRIA II beads; thus the specificity of the RIA was also improved. That is, not only did the assay detect more antigen as shown by the cpm bound but the signal to noise ratio was also enhanced (calculated by dividing the cpm bound by background cpm of the negative control); the experimental data indicates that the sensitivity of the assay has been increased as well.

A reverse sandwich assay using (1) a total monoclonal system or (2) a combination of IgM and IgG monoclonal anti-HBs antibodies with the AUSRIA II reagents was then prepared. The incubation conditions were identical for all assays and therefore, the results are directly comparable.

A HBsAg standard was first incubated at 10 ng/ml with ¹²⁵ I-anti-HBs (guinea pig antisera AUSRIA II) or monoclonal IgG ¹²⁵ I-anti-HBs of varying specific activities (0.1 to 16 μCi/μg) for 1 hour at 45° C. Beads were washed and counted. The results are depicted in Table III.

                                      TABLE III                                    __________________________________________________________________________     Sensitivity of a 2 hr reverse monoclonal anti-HBs sandwich RIA for             HBsAg and improvement of AUSRIA II reagents by substitution with               monoclonal anti-HBs antibodies.                                                       cpm**                                                                              cpm cpm+   cpm                                                      Monoclonal*                                                                           5D3 neg.                                                                               AUSRIA II                                                                             neg.                                                                               S/N S/N                                              antibody                                                                              beads                                                                              cont.                                                                              beads  cont.                                                                              5D3 AUSRIA II                                        __________________________________________________________________________     125    20,809                                                                             235 20,632 136 88.5                                                                               151.7                                            I-1C7-PA                                                                       125    5,138                                                                              50  5,672  40  113.4                                                                              141.8                                            I-1C7-J                                                                        125    19,131                                                                             214 16,697 321 89.4                                                                               52.0                                             I-1C7-C                                                                        125    15,619                                                                             196 14,916 340 79.7                                                                               43.8                                             I-4E8                                                                          125    3,634                                                                              26  1,987  21  139.8                                                                              94.0                                             I-anti-HBs                                                                     (AUSRIA II)                                                                    __________________________________________________________________________      *1C7-PA, 1C7J, and 1C7C are IgG, monoclonal antiHBs antibodies labeled         with different specific activities. These antibodies were incubated with       10 ng/ml of HBsAg standard for 1 hr at 45° C.                           **5D3, an IgM monoclonal antiHBs, was coated to beads and incubated for a      second 1 hr period. Washed ×3 and counted. +AUSRIA II beads added a      the second component instead of 5D3 coated beads.  AUSRIA II run in the        forward direction (e.g. beads first incubated for 1 hr at 45° C.        with HBs Ag standard, then washed, and 200 μl .sup.125 Ianti-HBs            (guinea pig) added for an additional 1 hr period) yields a S/N of 20.6.  

It is noteworthy that the sensitivity of the RIAs has been improved under all experimental conditions: further the total monoclonal sandwich assay was very sensitive for detection of HBsAg. In addition, the AUSRIA II assay was also substantially improved by the use of ¹²⁵ I-IgG monoclonal anti-HBs or by substituting the 5D3 coated beads for their beads in the reverse direction assay. These studies establish the value of monoclonal anti-HBs antibodies as a sensitive and specific probe either alone or in combination with the AUSRIA II or the like reagents in the improvement of immunoassays for HBsAg.

Investigations were performed to determine the sensitivity by a quantitative titration of HBsAg standards as shown in Table IV.

                                      TABLE IV                                     __________________________________________________________________________     Development of a more sensitive 2 hour reverse sandwich monoclonal             RIA for HBsAg with a quantitative comparison to the AUSRIA II RIA                     Conc.                                                                              Mean   Mean                                                                HBsAg                                                                              cpm*   cpm** S/N+   S/N                                             Dilution                                                                              ng/ml                                                                              AUSRIA II                                                                             5D3 + 1C7                                                                            AUSRIA II                                                                             5D3 + 1C7                                       __________________________________________________________________________     Undiluted                                                                             20  4,864  19,992                                                                               94.8   995.5                                           1:2    10  2,098  608   27.9   30.0                                            1:4    5   972    397   14.9   20.0                                            1:8    2.5 560    180   8.60   9.0                                             1:16   1.25                                                                               279    164   4.41   8.2                                             1:32   .625                                                                               62     98    .95    4.9                                             1:64   .312                                                                               81     42    1.2    2.0                                             Neg. controls                                                                             65     20                                                           __________________________________________________________________________      *Assay performed by incubating AUSRIA II beads ×1 hour at 45.degree      C. with 200 μl of HBsAg standards. Beads were washed ×3 followed      by the addition of 200 μl .sup.125 Ianti-HBS (AUSRIA II: 166,642 cpm)       for an additional 1 hr period. Beads washed and counted in a gamma             counter.                                                                       **Assay performed in the reverse direction where antigen standard (200         μl) added to 1C7 .sup.125 IIgG.sub.1 monoclonal antiHBs (150,000 cpm)       for 1 hour at 45% C. Then 5D3 coated beads were added for an additional 1      hour period. Beads were washed and counted as above. +S/N represents           signal to noise ratio by dividing mean cpm of test sample by mean cpm of       control. Note that the total monoclonal reverse sandwich assay detected        HBsAg at a concentration of 0.301 ng/ml.                                 

In this experiment a total monoclonal assay was compared with AUSRIA II; both assays were run in the reverse direction as described above. AUSRIA II detected HBsAg at a level of 1.25 ng/ml in the reverse direction as described above. AUSRIA II detected HBsAg at a level of 1.25 ng/ml in the reverse direction (lowest level of sensitivity was 2.5 ng/ml in the forward direction.). A combination of 5D3 coated beads with monoclonal 125I-IgG anti-HBs (1C7), however detected HBsAg at 0.3012 ng/ml which represent an approximate 4-fold increase in sensitivity. The data suggest again that a monoclonal IgM and IgG system was superior to conventional assays.

An assay run was developed in the forward direction either alone or in combination with AUSRIA II reagents. Table V illustrates the results of these experiments.

                                      TABLE V                                      __________________________________________________________________________     Development of a monoclonal assay for HBsAg in the forward direction           with                                                                           comparison to the AUSRIA II reagents.                                                 Mean*  Mean** Mean+     Mean                                            Serum  cpm    cpm    cpm       cpm                                             dilution                                                                              5D3 + 1 C7                                                                            AUSRIA II                                                                             AUSRIA II + 1C7                                                                          5D3 + AUSRIA II                                 __________________________________________________________________________     Undiluted                                                                             9,260  8,924  10,117    6,184                                           1 × 10.sup.1                                                                    6,251  4,621  9,162     3,215                                           1 × 10.sup.2                                                                    3,418  1,151  7,164     2,001                                           1 × 10.sup.3                                                                    2,969  962    6,218     943                                             1 × 10.sup.4                                                                    2,169  213    5,164     512                                             Neg. controls                                                                         992    150    1,521     150                                             __________________________________________________________________________      *Serum dilutions incubated for 16 hrs with 5D3 coated beads. Beads washed      ×3 with distilled water. The second incubation was for 4 hrs with        .sup.125 I1C7 (160,000 cpm). Beads were washed and counted.                    **Standard AUSRIA II radioimmunoassay performed in an identical manner to      that mentioned above. +Identical radioimmunoassay but where .sup.125 I1C7      was substituted for .sup.125 Ianti-HBs (AUSRIA II)  Identical                  radioimmunoassay but where .sup.125 Ianti-HBs (AUSRIA II) was substituted      for .sup.125 I1C7 and AUSRIA II beads were substituted for 5D3 coated          beads.                                                                   

All assays were performed under the following conditions: HBsAg positive dilutions(200 μl) were incubated with 5D3 or AUSRIA II beads for 16 hours at room temperature. The beads were washed X3 with distilled water. The second incubation covered 4 hours at room temperature with either monoclonal ¹²⁵ I-anti-HBs (AUSRIA II) probes. Tables V and VI demonstrate that very sensitive RIA's performed in the forward and reverse directions were developed using either a total monoclonal IgG-IgM or IgM-IgM system or a combination assay of monoclonal antibodies with AUSRIA II reagents.

                                      TABLE VI                                     __________________________________________________________________________     Development of a total IgM monoclonal anti HBs reverse sandwich                assay demonstrating enhanced sensitivity compared to AUSRIA II                 Dilution                                                                       HBsAg  Conc. HBsAg                                                                           cpm* bound cpm** bound                                           Standard                                                                              (ng/ml)                                                                               5D3 + 5D3                                                                             S/N AUSRIA II                                                                              S/N                                           __________________________________________________________________________     1:2    10     1010   6.97                                                                               3398    26.14                                         1:4    5      1449   9.99                                                                               793     6.10                                          1:8    2.5    1457   10.04                                                                              404     3.10                                          1:16   1.25   1713   11.82                                                                              256     1.96                                          1:32   0.625  1800   12.42                                                                              183     1.40                                          1:64   0.312  1749   12.06                                                                              205     1.57                                          1:128  0.156  1415   9.75                                                                               223     1.71                                          1:256  0.078  687    4.74                                                                               176     1.35                                          1:512  0.039  116    0.80                                                                               156     1.20                                          Neg. controls                                                                         --     145        130                                                   __________________________________________________________________________      .sup.*125 I5D3 (IgM monoclonal antiHBs) first incubated with HBsAg             standards (200,000 cpm) for 1 hr at 45° C., then 5D3 coated beads       were added for a further 1 hr incubation at 45° C. Beads were           washed ×3 with distilled water and counted.                              **Standard AUSRIA RIA run in the forward direction under the same              incubation conditions as mentioned above.                                

EXAMPLE II

This example describes the use of monoclonal IgM anti-HBs in the biopurification of HBsAg from serum.

MATERIALS AND METHODS

Monoclonal IgM anti-HBs affinity columns were prepared by coupling 28 and 65 mg of ascites fluid protein derived from cell lines 5D3 and 2F11 respectively to cyanogen bromide activated Sepharose 6B; Williams et al, Cell 12:663 (1977). One control column was comprised of cyanogen bromide activated Sepharose 6B alone and the other was coupled to 30 mg of mouse serum. Human serum (10 mg) derived from a high titer chronic HBsAg (adw subtype) carrier patient was placed over all four columns and incubated for 24 hours at room temperature. The columns were extensively washed with PBS. Subsequently, 1 ml fractions were collected by elution with glycine-HCl buffer pH 2.6. The pH of each fraction was adjusted to 7.4 with 0.1N NaOH and the protein concentration, Lowry et al, J. Biol. Chem. 193:265 (1951) and HBsAg activity (AUSRIA II), was determined.

RESULTS

FIGS. 1-4 demonstrate that HBsAg (adw subtype) present in serum, bound to the monoclonal IgM anti-HBs affinity columns prepared from ascites fluid derived from clones 5D3 and 2F11. It is noteworthy that previous studies have established that monoclonal IgM anti-HBs from 2F11 and 5D3 recognized the adw subtype. Subsequently, the HBsAg could be recovered from the columns in μg amounts by elution with glycine HCl buffer pH 2.6. In contrast, there was no significant binding of HBsAg to the Sepharose 6B column or to Sepharose 6B coupled to mouse serum. This study illustrates the use of monoclonal IgM in the purification and characterization of antigens.

EXAMPLE III

This example describes the use of monoclonal IgM antibodies in the detection of HBsAg coated to red blood cells. The experiments demonstrate that is also feasible to detect free HBsAg in serum by first coating IgM anti-HBs to red blood cells and incubating such cells with HBsAg-containing serum to produce a hemagglutination reaction.

MATERIALS AND METHODS

Human O-negative red blood cells coated with HBsAg subtypes adw and ayw were purchased commercially. 25 μl of buffer was placed in microtiter plates followed by serial 2-fold dilution of culture supernatants and ascites fluid from three IgM anti-HBs producing hybridoma cell lines: plates were incubated at 37° C. for 30 minutes. Following this incubation 25 μl of a 0.1% solution of HBsAg coated cells were added to each well. The reaction mixture was allowed to incubate at 37° C. for 30 minutes, plates were centrifuged at 300×g for 2 minutes and placed at a 45° angle. After 10 minutes the plates were assessed for the presence or absence of hemagglutination.

RESULTS

Studies were performed with HBsAg coated human O-negative red blood cells in order to determine if the monoclonal IgM anti-HBs antibodies possessed agglutinating properties and recognized subtypes of HBsAg as shown in Table VII.

                  TABLE VII                                                        ______________________________________                                         Hemagglutination titer of IgM anti-HBs for HBsAg (adw and ayw)                 coated human red blood cells                                                   Culture supernatants*                                                                             Ascites fluid*                                              Cell line                                                                              ayw      adw       ayw      adw                                        ______________________________________                                         1F8     1:4      1:2.6 × 10.sup.5                                                                   1:2.6 × 10.sup.4                                                                  1:1.7 × 10.sup.10                    2F11    1.4      1:1.3 × 10.sup.5                                                                   1:1 0 × 10.sup.6                                                                  1:1.3 × 10.sup.11                    5D3     1:2.1 ×                                                                           1:3.2 × 10.sup.7                                                                   1:2.2 × 10.sup.12                                                                 1:2.7 × 10.sup.11                            10.sup.9                                                               Controls                                                                               <1:4     <1:4      <1:4     <1:4                                       ______________________________________                                          *Hemagglutination titer of HBsAg coated human O negative red blood cells       HBsAg (subtypes adw and ayw). Positive titers are considered 1:4 or            greater. Data are expressed as the final dilutions of the original 25          μl of ascites fluid and cell culture supernatants which gave a              hemagglutination reaction.  Controls consist of ascites fluid containing       IgG.sub.1 monoclonal antibodies to cardiac myosin, reacting with HBsAg         coated cells. Ascites fluid from the above three cloned cell lines             reacting with human O negative red blood cells (HBsAg negative) and mouse      myeloma IgM 1 mg/ml in phosphate buffered saline reacting with HBsAg           coated red blood cells and control red blood cells.                      

It is of great interest that these IgM anti-HBs are exceptionally good agglutinating antibodies. For example 25 μl of cell supernatant from line 5D3 gave hemagglutination titers of 1:2.1×10⁹ and 1:3.2×10⁷ for HBsAg (ayw and adw) coated red blood cells respectively. Further, the agglutinating capacity was amplified in the ascites fluid since extraordinary high titers of 1:2.2×10¹² and 1:2.7×10¹¹ were noted for the same HBsAg coated cells. Indeed it is remarkable that 25 μl of ascites fluid diluted at 1:2.2×10¹² still gave a positive agglutination reaction and illustrates the apparent high avidity of this IgM anti-HBs for HBsAg coupled indicator cells. Thus, the above data indicates that monoclonal IgM anti-HBs is an extremely potent antibody for hemagglutination reactions, and is of considerable use in the detection of HBsAg in serum by first coating the antibody to red blood cells and reacting such cells with HBsAg positive serum.

EXAMPLE IV

In this example, it is demonstrated that monoclonal IgM anti-HBs may be employed as a probe for detection of anti-HBs in serum by an inhibition radioimmunoassay.

MATERIALS AND METHODS

In this assay μl of human serum containing anti-HBsAg (5 ng/ml) followed by the addition of monoclonal ¹²⁵ I-1C7 (200,000 cpm) and incubated for 1 hour at 45° C. The beads were washed x3 with distilled water and counted in a gamma well counter.

RESULTS

Table VIII illustrates the results of the experiment.

                  TABLE VIII                                                       ______________________________________                                         Development of a monoclonal                                                    radioimmunoassay for anti-HBs in serum by inhibitor of                         antigen (HBsAg) binding                                                        Serum dilution*                                                                              mean cpm** bound                                                 ______________________________________                                         Undiluted     106                                                              1 × 10.sup.1                                                                           131                                                              1 × 10.sup.2                                                                           492                                                              1 × 10.sup.3                                                                           1,062                                                            1 × 10.sup.4                                                                           2,615                                                            Neg controls X                                                                               98                                                               ______________________________________                                          *Patient serum containing antiHBs.                                             **The assay was performed as follows: serum containing antiHBs (bound          15,612 cpm in AUSAB radioimmunoassay) was added to a fixed concentration       of unlabeled HBsAg followed by the addition of .sup.125 I1C7 (monoclonal       IgG.sub.1 antiHBs) and incubated for 1 hr. at 45° C. Next 5D3           coated beads (IgM monoclonal antiHBs) were added to the reaction mixture       for 1 hr., washed ×3 and counted in a Packard gamma counter.             Antibody to hepatitis B surface antigen (antiHBs) in serum complexed with      added HBsAg and therefore was unavailable for binding with .sup.125 I1C7       monoclonal antiHBs at undiluted and a 1:10 serum dilution. However, as th      concentration of antiHBs becomes lower as the result of further dilution,      the HBsAg now becomes available for binding to .sup.125 I1C7 antiHBs.          Thus, free HBsAg was now in excess of antiHBs in serum and was detectable      in the RIA with increasing serum dilution. This assay, therefore, measure      inhibition of antigen binding by serum containing antiHBs and is a             sensitive method for detection of such antibodies.                       

The number of counts of ¹²⁵ I-1C7 (monoclonal IgG₁ anti-HBs) bound to the beads increased with progressive serum dilution. This binding results from the fact that antibody to hepatitis B surface antigen (anti-HBs) in test serum complexes first to HBsAg, and therefore was unavailable for binding with the radiolabeled probe ¹²⁵ I-1C7) at both the undiluted and 1:10 serum dilution. However, as the concentration of anti-HBs in serum becomes lower as the result of further dilution, the added HBsAg (5 ng/ml) now becomes available for binding to ¹²⁵ I-1C7 anti-HBs. Thus, HBsAg is now in excess of anti-HBs concentrations in serum at the higher dilutions and is detectable as shown by increasing cpm bound to the 5D3 coated beads in the RIA. This assay therefore measures inhibition of antigen binding (HBsAg) by serum containing anti-HBs and is a sensitive method for detection of such antibodies.

COMPARATIVE EXAMPLE

The affinity of anti-HBsAg monoclonal IgM for HBsAg was measured by Scatchard analysis and the results are as follows:

    ______________________________________                                         Antibody         K.sub.a (M.sup.-1)                                                                            Ref                                            ______________________________________                                         Hybridomally produced IgM                                                      of present invention                                                                            0.048 - 4.1 × 10.sup.11                                                                 invention                                      against HBsAg                                                                  ______________________________________                                          The affinity constants expressed in this table and elsewhere in this           application and claims refer to binding affinities per molecule of IgM an      should not be construed to represent affinities per binding site of IgM. 

Cell line 5D3, producing the monoclonal IgM anti-HBs described in the examples, was deposited at the American Type Culture Collection, Rockville, Md., on Aug. 25, 1988, under the Budapest Treaty, and given ATCC Accession No. HB9801. 

What is claimed as new and desired to be secured by Letters Patent of the United States is:
 1. An immunoassay method utilizing an antibody to assay for a substance comprising hepatitis B-surface antigen (HBsAg) determinants which comprises the steps of:contacting a test sample containing said substance comprising HBsAg determinants with said antibody; and determining the presence of said substance in said sample; wherein said antibody is a monoclonal high affinity IgM antibody having a binding affinity constant for said HBsAg determinants of at least 10⁹ M⁻¹.
 2. The assay method of claim 1 wherein said constant is at least 10¹⁰ M⁻¹.
 3. The assay method of claim 1 which is selected from the group consisting of radioimmunoassay, enzyme immunoassay, agglutination immunoassay, and fluorescence immunoassay.
 4. The assay method of claim 3 which is radioimmunoassay.
 5. The method of claim 1 wherein said HBsAg determinants are present in Hepatitis B virus.
 6. The assay method of claim 1 wherein said monoclonal high affinity IgM antibodies are coupled to an insoluble solid phase.
 7. Monoclonal high affinity IgM antibodies immunoreactive with HBsAg determinants wherein said antibodies are coupled to an insoluble solid phase, and wherein the binding affinity constant of said antibodies for said HBsAg determinants is at least 10⁹ M⁻¹.
 8. The antibodies of claim 7 which are immunoreactive with hepatitis B surface antigen.
 9. The antibodies of claim 8 wherein said label is a radiolabel. 